The invention relates to anti-thiol reagents which inhibit enzyme activity of cell-associated protein disulfide isomerase (PDI) by oxidizing or blocking PDI active site vicinal thiol groups which normally participate in disulfide bond rearrangement of PDI substrates. Inhibition of this PDI function is particularly useful in blocking PDI-mediated entry of HIV or other virions into a host cell.
The present invention also relates to anti-inflammatory agents.
The invention further relates to an assay for the identification of such PDI inhibitors based on the discovery that inhibitors of the invention also induce shedding of the leukocyte L-selectin adhesion molecule.
The invention further relates to the use of calmodulin antagonist reagents to induce L-selectin shedding.
1. Field of Art
PDI (protein disulfide isomerase) is a constitutive intracellular protein that is also found to be expressed on the surface of many mammalian cell types, including immune system cells, hepatocytes, and platelets. Like other members of the thyredoxin superfamily of proteins, PDI is a multifunctional redox-sensitive protein that catalyzes oxidation-reduction reactions via a vicinal dithiol-dependent disulfide-sulfhydryl interchange between its internal vicinal dithiol (Cys-Gly-His-Cys) active sites and the disulfide bonds of its substrates to promote their reconfiguration. PDI recognizes the side chains of cysteine residues in its substrates, and it is its two vicinal dithiol groups, two on each of two identical PDI subunits, that are critical for its enzymatic isomerase function, in particular its broad specificity for correcting the configuration of a large spectrum of proteins as needed. For example, PDI is present in the endoplasmic reticulum of most cells, where it is believed to mediate co- and post-translational modifications of nascent proteins with incorrect sulfide bonds; it is also present in certain protein complexes such as triglyceride transfer protein complex (MTP) wherein it maintains the complex in a catalytically-active state and inhibits complex aggregation. Membrane PDI catalyzes the cleavage of disulfide bonds during the earliest stages of endocytosis, and activates diphtheria toxin by catalyzing cleavage of this disulfide-linked dimer. PDI also catalyzes the isomerization of thrombospondin (TSP) disulfide bonds, thereby profoundly modulating TSP-ligand binding activity. Both TSP and PDI are released by activated platelets; PDI is also released by degranulated neutrophils (J. Cell Physiol. 144: 280, 1990).
Other known PDI functions include the recognized ability of PDI to modulate certain adhesive interactions. While PDI isomerase activity affects, for example, the adhesive properties of TSP, PDI is additionally a “chaperone” for some proteins by means independent of its catalytic activity. One of these chaperone functions has been attributed to PDI binding in a complex formation with proteins which have a tendency to aggregate in the denatured state. Association with PDI prevents this aggregation by promoting appropriate folding of the associated protein. PDI in MTP complexes inhibits MTP aggregation, and a PDI homolog (cognin) plays a role in the adhesion-dependent aggregation of retinal cells.
2. Discussion of Related Art
Of particular relevance to the present invention is the involvement of PDI in the shedding of the human thyrotropin (TSH) receptor ectodomain (Biochem, 35:14800, 1996). In a two-step process, a matrix metalloproteinase first cleaves the receptor into two subunits (an α-extracellular subunit and a β-transmembrane subunit) linked by a disulfide bridge. The α-extracellular subunit is then shed from the cell membrane as a result of PDI-mediated reduction of the disulfide bridge(s) connecting it to the β-transmembrane subunit. However, in contrast to the PDI-mediated L-selectin shedding mechanism according to the present invention, the TSH shedding mechanism requires PDI isomerase activity, and inhibition of PDI activity with known PDI inhibitors such as DTNB (5,5′-dithiobis (2-nitrobenzoic acid), bacitracin, or anti-PDI antibodies to prevent the shedding (release) of the TSH α-subunit.
Also of relevance is the known ability of PDI to mediate transmembrane carriage of proteins and virions into cells by rearrangement of their disulfide bonds. For example, the attachment of HIV to its host cell surface receptor CD4 via the viral glycoprotein gp120 triggers a conformational change in gp120/gp 41 resulting from a rearrangement of its critical disulfide bonds as catalyzed by PDI. Known PDI inhibitors (e.g., bacitracin, anti-PDI antibodies) block HIV entry into the cell cytoplasm to some extent, but they are very weak inhibitors of PDI isomerase activity in this clinical application (PNAS USA 91: 4559, 1994). The use of another known PDI inhibitor, DTNB (supra) to inhibit viral penetration into cells has been described (U.S. Pat. No. 5,532,154 to Brown); however, the recited activity of this compound in preventing HIV entry into cells is attributed by the patentee to inactivation of “virus-derived thiol reductase/protein disulfide isomerase”, presumably encoded by and present on the virus itself.
The interaction of arsine oxide with certain proteins having active vicinal dithiol sites which undergo catalytic conversion to disulfides to form stable dithioarsenic derivatives is described in Anal. BioChem 212: 325-334 (1993). This reactivity was used by the authors to separate dithiols from monothiols and also from dithiol-containing proteins with low-affinity for arsine oxide.